Fluorescence-based sensor longevity—the period during which sensors exhibit reproducible responses when exposed to an analyte—is limited. As the sensors are repeatedly exposed to light over time, they become less responsive, typically due to photobleaching. Certain sensor arrays retain responsiveness for an average of approximately one hundred responses or a total exposure time of less than 8 minutes.
The limited sensor life can be a limiting factor in the use of sensor arrays. In some cases, replacement of non-responsive sensors means having to replace the entire array or some portion thereof.
Sensor arrays for analyzing vapors (also referred to as “electronic noses”), for example, are limited by sensor life. Electronic noses detect changes in the response patterns of non-specific sensors exposed to vapors and categorize the vapors with the use of pattern recognition software. Ideally, electronic noses contain a diverse array of cross-reactive sensors. These arrays are typically composed of conducting polymer sensors or fluorescent sensors that produce patterns resulting from changes in the properties of the sensors over time. Such arrays use pattern recognition, storing the response patterns in a large database such that a wide range of vapors can subsequently be identified using the database. That is, during the training process, responses are collected from known vapors and those responses are then used to classify unknown vapors. A training database can be collected on one array and applied to multiple arrays over time, which eliminates the need for repetitive training. Ideally, a database should contain many replicate array responses from many vapors. One of the most important requirements for building large databases of vapor patterns is array longevity. In fact, for many such applications, only sensor arrays with long-term use capability and with less frequent replacement and servicing needs will be acceptable. It is understood that the more analytes that can be recognized, the more effective the array, thereby underscoring the need for an increased number of training replicates and inclusion of additional vapors in the training database. If an array degrades too fast, it will have to be replaced and the training will have to be repeated. Thus, the longevity of most fluorescent sensor arrays has typically been limited by the photobleaching rates of the fluorescent dyes attached to the individual beads.
Several approaches to overcome or compensate for photobleaching of fluorescent dyes and fluorescent sensors have focused on the dyes or sensing substrates. For example, certain ratiometric sensors have been introduced that are insensitive to photobleaching effects. See Kermis, H. R.; Kostov, Y.; Harms, P.; Rao, G. Biotechnol. Progress 2002, 18, 1047-1053; Song, A.; Parus, S.; Kopelman, R. Anal. Chem. 1997, 69, 863-867; and Xu, Z.; Rollins, A.; Alcala, R.; Marchant, R. E. J. Biomed. Mater. Res. 1998, 39, 9-15. Further, anti-fading agents have been added to dyes to chemically decrease the photobleaching rates of the dyes. See Berrios, M.; Conlon, K. A.; Colflesh, D. E. Methods in Enzymology 1999, 307, 55-79; Krenik, K. D.; Kephart, G. M.; Offord, K. P.; Dunnette, S. L.; Gleich, G. J. Journal of Immunological Methods 1989, 117, 91-97. Another approach involves increasing dye photostability by engineering fluorescent polymer molecules with structural properties that prevent π-stacking, thereby circumventing the fluorescence quenching caused by such π-stacking. See Yang, J.-S.; Swager, T. M. J. Am. Chem. Soc. 1998, 120, 5321-5322. Although somewhat successful, each of the above methods requires chemical modification of the indicator dyes or sensing substrates, or, in the case of ratiometric measurements, the use of different indicators altogether.
Other approaches have taken advantage of the physical properties of sensors to increase sensor longevity. One such approach involves incorporating fluorescent dye into a polymer matrix, resulting in a polymer layer containing freely diffusible dye molecules. See Shortreed, M., Monson, E., and R. Kopelman, Anal. Chem. 1996, 68, 4015-4019; Barnard, S. M. and D. R. Walt, “Chemical Sensors Based on Controlled-Release Polymer Systems,” Science, 1991, 251 (4996): 927-9; Agayn, V. and D. R. Walt, “Fiber-Optic Immunosensors Based on Continuous Reagent Delivery,” Immunomethods, 1993, 3(2): 112-21; and Uttamlal, M. and D. R. Walt, “A Fiber-Optic Carbon Dioxide Sensor for Fermentation Monitoring,” Bio/Technology, 1995, 13 (6): 597-601. In use, a small portion of the layer is selectively illuminated and as the dye molecules are photobleached, they are replaced with new dye molecules that diffuse from a reservoir into the illuminated polymer film area, thereby increasing the number of sensor measurements possible. Another approach involving an IR detector focused on sectioning the array with an optical slit. See Takayama, R.; Tomita, Y.; Asayama, J.; Nomura, K., and H. Ogawa, Sens. Actuators A: Physical 1990, A22, 508-512. That is, the optical slit served to selectively illuminate individual IR detectors in a microarray and the response of each detector was monitored during and after the illumination.
However, the Shortreed et al. and Walt et al. methods described above are limited to devices using the polymer layer containing diffusible molecules. Thus, the methods cannot be implemented using any known non-diffusible sensors. Further, the Takayama et al. optical slit described above is used in conjunction with an array comprised of only one type of sensor.
There is a need in the art for a method and system of array imaging that extends or maximizes the longevity of the sensor array by minimizing the effects of photobleaching.